Loss-of-Function Screen Reveals Novel Regulators Required forDrosophilaGermline Stem Cell Self-Renewal

Abstract

The germline stem cells (GSCs) of Drosophila melanogaster ovary provide an excellent model system to study the molecular mechanisms of stem cell self-renewal. To reveal novel factors required for Drosophila female GSC maintenance and/or division, we performed a loss-of-function screen in GSCs by using a collection of P-element–induced alleles of essential genes. Mutations in genes of various functional groups were identified to cause defects in GSC self-renewal. Here we report that a group of mutations affecting various ubiquitin-conjugating enzymes cause significant GSCs loss, including Plenty of SH3s (POSH), Ubiquitin-conjugating enzyme 10 (UbcD10), and pineapple eye (pie). Ubiquitin-mediated protein degradation plays a variety of roles in the regulation of many developmental processes, including mediating stem cell division through degradation of cell cycle regulators. We demonstrated that pie, sharing highly conserved RING domains with human E3 ubiquitin ligase G2E3 that are critical for early embryonic development, is specifically required for GSC maintenance, possibly through regulation of bone morphogenetic protein signaling pathway. Despite the previously reported role in imaginal disc cell survival, pie loss-of-function induced GSC loss is not to the result of caspase-involved cell death. Further efforts are needed to elucidate the functions of ubiquitin ligases in GSC maintenance, which will ultimately contribute to a better understanding of how the ubiquitin-conjugating enzymes regulate stem cell biology in mammalian systems.

Tie-mediated signal from apoptotic cells protects stem cells in Drosophila melanogaster

“…2f,h; Supplementary Table 4). This is in agreement with previous observations that loss of pie in GSCs does not induce apoptosis 22 . However, esgGal4 driven pie RNAi expression did lead to a significant reduction of the esg + cells, which was not rescued by the co-expression of the caspase inhibitor p35 (Fig.…”

“…A previous screen for essential factors in stem cell self-renewal identified a cell survival factor, pie 22 . As the Drosophila homologue of human G2E3 ubiquitin ligase, pie was originally reported to be required for cell survival in the developing Drosophila eyes 23,24 .…”

Loss of foxo rescues stem cell aging in Drosophila germ line

“…We were able to identify critical roles for two known proteins involved in tissue homeostasis: Foxo for cell cycle withdrawal and Tor for cell cycle reentry. foxo has been well documented as a regulator of stem cell self-renewal and quiescence ( Demontis and Perrimon, 2010 ; Xing et al, 2012 ; Eijkelenboom and Burgering, 2013 ; Gopinath et al, 2014 ; Xing et al, 2015 ). Notably, foxo tends not to be active during normal physiology, but rather during stressful conditions, when it responds to and counteracts a stressor in order to maintain homeostasis ( Kenyon, 2010 ; Eijkelenboom and Burgering, 2013 ).…”

Local Cell Death Changes the Orientation of Cell Division in the Developing Drosophila Wing Imaginal Disc Without Using Fat or Dachsous as Orienting Signals

“…This provides a useful assay for signals produced by dying cells, since the rate of apoptosis is not sufficient to eliminate the pie mutant clones completely, which provide a continuous source of dying cells. The pie gene is also required for germline stem cell self-renewal, where it may affect BMP signaling[45]. …”